Cell differentiation is the central characteristic of tissue morphogenesis which initiates during embryogenesis, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.
The cellular and molecular events which govern the stimulus for differentiation of cells is an area of intensive research. In the medical and veterinary fields, it is anticipated that the discovery of the factor or factors which control cell differentiation and tissue morphogenesis will advance significantly medicine's ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas for human and veterinary therapeutics include reconstructive surgery and in the treatment of tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative nerve diseases, inflammatory diseases, and cancer, and in the regeneration of tissues, organs and limbs. (In this and related applications, the terms “morphogenetic” and “morphogenic” are used interchangeably.)
A number of different factors have been isolated in recent years which appear to play a role in cell differentiation. Recently, a distinct subfamily of the “superfamily” of structurally related proteins referred to in the art as the “transfoming growth factor-b (TGF-β) superfamily of proteins” have been identified as true tissue morphogens.
The members of this distinct “subfamily” of true tissue morphogenic proteins share substantial amino acid sequence homology within their morphogenetically active C-terminal domains (at least 50% identity in the C-terminal 102 amino acid sequence), including a conserved six or seven cysteine skeleton, and share the in vivo activity of inducing tissue-specific morphogenesis in a variety of organs and tissues. The proteins apparently contact and interact with progenitor cells e.g., by binding suitable cell surface molecules, predisposing or otherwise stimulating the cells to proliferate and differentiate in a morphogenetically permissive environment. These morphogenic proteins are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve innervation as required by the naturally occurring tissue. The proteins have been shown to induce morphogenesis of both bone cartilage and bone, as well as periodontal tissues, dentin, liver, and neural tissue, including retinal tissue.
The true tissue morphogenic proteins identified to date include proteins originally identified as bone inductive proteins. These include OP-1, (osteogenic protein-1, also referred to in related applications as “OP1”), its Drosophila homolog, 60A, with which it shares 69% identity in the C-terminal “seven cysteine” domain, and the related proteins OP-2 (also referred to in related applications as “OP2”) and OP-3, both of which share approximately 70-75% identity with OP-1 in the C-terminal seven cysteine domain, as well as BMP5, BMP6 and its murine homolog, Vgr-1, all of which share greater than 85% identity with OP-1 in the C-terminal seven cysteine domain, and the BMP6 Xenopus homolog, Vg1, which shares approximately 57% identity with OP-1 in the C-terminal seven cysteine domain. Other bone inductive proteins include the CBMP2 proteins (also referred to in the art as BMP2 and BMP4) and their Drosophila homolog, DPP. Another tissue morphogenic protein is GDF-1 (from mouse). See, for example, PCT documents US92/01968 and US92/07358, the disclosures of which are incorporated herein by reference.
As stated above, these true tissue morphogenic proteins are recognized in the art as a distinct subfamily of proteins different from other members of the TGF-β superfamily in that they share a high degree of sequence identity in the C-terminal domain and in that the true tissue morphogenic proteins are able to induce, on their own, the full cascade of events that result in formation of functional tissue rather than merely inducing formation of fibrotic (scar) tissue. Specifically, members of the family of morphogenic proteins are capable of all of the following in a morphogenetically permissive environment: stimulating cell proliferation and cell differentiation, and supporting the growth and maintenance of differentiated cells. The morphogenic proteins apparently may act as endocrine, paracrine or autocrine factors.
The morphogenic proteins are capable of significant species “crosstalk.” That is, xenogenic (foreign species) homologs of these proteins can substitute for one another in functional activity. For example, DPP and 60A, two Drosophila proteins, can substitute for their mammalian homologs, BMP2/4 and OP-1, respectively, and induce endochondral bone formation at a non-bony site in a standard rat bone formation assay. Similarly, BMP2 has been shown to rescue a dpp mutation in Drosophila. In their native form, however, the proteins appear to be tissue-specific, each protein typically being expressed in or provided to one or only a few tissues or, alternatively, expressed only at particular times during development. For example, GDF-1 appears to be expressed primarily in neural tissue, while OP-2 appears to be expressed at relatively high levels in early (e.g., 8-day) mouse embryos. The endogenous morphogens may be synthesized by the cells on which they act, by neighboring cells, or by cells of a distant tissue, the secreted protein being transported to the cells to be acted on.
A particularly potent tissue morphogenic protein is OP-1. This protein, and its xenogenic homologs, are expressed in a number of tissues, primarily in tissues of urogenital origin, as well as in bone, mammary and salivary gland tissue, reproductive tissues, and gastrointestinal tract tissue. It is also expressed in different tissues during embryogenesis, its presence coincident with the onset of morphogenesis of that tissue.
The morphogenic protein signal transduction across a cell membrane appears to occur as a result of specific binding interaction with one or more cell surface receptors. Recent studies on cell surface receptor binding of various members of the TGF-β protein superfamily suggests that the ligands can mediate their activity by interaction with two different receptors, referred to as Type I and Type II receptors to form a hetero-complex. A cell surface bound beta-glycan also may enhance the binding interaction. The Type I and Type II receptors are both serine/threonine kinases, and share similar structures: an intracellular domain that consists essentially of the kinase, a short, extended hydrophobic sequence sufficient to span the membrane one time, and an extracellular domain characterized by a high concentration of conserved cysteines.
A number of Type II receptor sequences recently have been identified. These include “TGF-βR II”, a TGF-β Type II receptor (Lin et al. (1992) Cell 68:775-785); and numerous activin-binding receptors. See, for example, Mathews et al. (1991) Cell 65:973-982 and international patent application WO 92/20793, published Nov. 26, 1992, disclosing the “ActR II” sequence; Attisano et al., (1992) Cell 68:97-108, disclosing the “ActR-IIB” sequence; and Legerski et al. (1992) Biochem Biophys. Res. Commun 183:672-679. A different Type II receptor shown to have affinity for activin is Atr-II (Childs et al. (1993) PNAS 90:9475-9479.) Two Type II receptors have been identified in C. elegans, the daf-1 gene, (Georgi et al. (1990) Cell 61:635-645), having no known ligand to date, and daf-4, which has been shown to bind BMP4, but not activin or TGF-β (Estevez, et al. (1993) Nature 365:644-649.)
Ten Dijke et al. disclose the cloning of six different Type I cell surface receptors from murine and human cDNA libraries. ((1993) Oncogene 8:2879-2887, and Science (1994) 264:101-104. These receptors, referenced as ALK-1 to ALK-6 (“activin receptor-like kinases”), share significant sequence identities (60-79%) and several have been identified as TGF-β binding (ALK-5) or activin binding (ALK-2, ALK-4) receptors. Xie et al. also report a Drosophila Type I receptor encoded by the sax gene (Science (1994) 263:1756-1759). The authors suggest that the protein binds DPP.
To date, the Type I receptors with which the morphogenic proteins described herein interact on the cell surface have not yet been identified, and no Type II receptor has been described as having binding affinity for OP-1 and its related sequences. Identification of these cell surface molecules, with which the morphogens interact and through which they may mediate their biological effect, is anticipated to enhance elucidation of the molecular mechanism of tissue morphogenesis and to enable development of morphogen receptor binding “analogs”, e.g., compounds (which may or may not be amino acid-based macromolecules) capable of mimicing the binding affinity of a morphogen for its receptor sufficiently to act either as a receptor binding agonist or antagonist. These “analogs” have particular utility in therapeutic, diagnostic and experimental research applications.
It is an object of this invention to provide nucleic acid molecules and amino acid sequences encoding morphogenic protein binding cell surface receptors, particularly OP-1-specific binding receptor sequences. Another object is to provide methods for identifying genes in a variety of species and/or tissues, and in a variety of nucleic acid libraries encoding morphogenic protein binding receptors, particularly receptors that bind OP-1. Still another object is to provide means for designing biosynthetic receptor-binding ligand analogs, particularly OP-1 analogs, and/or for identifying natural-occurring ligand analogs, including agonists and antagonists, using the receptor molecules described herein, and analogs thereof. Another object is to provide antagonists, including soluble receptor constructs comprising the extracellular ligand-binding domain, which can modulate the availability of OP1 for receptor binding in vivo. Another obect is to provide means and compositions for competing with activin-receptor and BMP2/4-receptor interactions. Yet another object is to provide means and compositions for ligand affinity purification and for diagnostic detection and quantification of ligands in a body fluid using OP1-specific cell surface receptors and ligand-binding fragments thereof. Still another object is to provides means and compositions for modulating the endogenous expression or concentration of these receptor molecules. Yet another object is to provide ligand-receptor complexes and analog sequences thereof, as well as antibodies capable of identifying and distinguishing the complex from its component proteins. Still another object is to provide means and compositions for modulating a morphogenesis in a mammal. These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.